We propose to pursue our research toward the development of an immunoassay incorporating laser fluorimetry. We will assay for the protein hormone insulin via an enzyme-linked procedure. The label employed is beta-galactosidase, which converts the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside to fluorescein. We will measure the fluorescein concentration by laser fluorimetry and thereby indirectly measure the enzyme activity. We will also develop a sandwich immunoassay for insulin. Fluorescein labeled antibody will bind to insulin bound to a solid phase antibody. After washing, fluorescein-Fc fragments will be released from the antibody by the addition of papain, and their concentration will be measured by laser fluorimetry. Finally, we will study the feasibility of laser-induced multiphoton ionization followed by mass spectrometry as a possible replacement for gas chromatography/mass spectrometry. We will first examine the aromatic molecule azulene (molecular weight 128).